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Rev. Col. Anest. 36: 11-18, 2008
ARTÍCULO DE invESiTigACión báSiCA
Molecular Diagnostics of Porcine Stress
Syndrome Susceptibility Associated with the
Arg615Cys Mutation Using Real-Time PCR with
Fluorescent Hybridization Probes
Jesús E. Rojas(1)*, Miriam A. Wilches(1)*, Libia A. Cepeda(1), María F. garcés(1), Miguel A.
Suarez(1), Rita M. baldrich(2), Cesar A. vélez(3), Mario F. guerrero(4), Martha R. garcía(5),
iván H. Moreno(5), Susana b. bravo(6), Radoslav Omelka(7) and Jorge E. Caminos(1)(6), §.
AbstrAct
Objective: The purpose of the present study was to develop a molecular genotyping method test by using
a real time PCR hybridization probe and applying it to the analysis of C1843T mutations of the Sus scrofa
RYR1 gene.
Animals population Three PSS-susceptible and PSS non-susceptible crossbred swine races were used for
the experiments: Pietrain X Landrace Belga, Pietrain X Large White and Pietrain X Duroc.
Methods: We have developed a genotyping method by using a hybridization probe and applied it to the
analysis of C1843T mutations of the RYR1 gene, associated with PSS susceptibility. Genotyping results
obtained by hybridization probe strategies were conirmed by restriction analysis and sequencing. In addition, phenotype/genotype correlation analyses were developed by using the in vitro contracture test and
conirmed the in vivo halothane-succinylcholine challenge.
results: The real-time PCR with luorescent hybridization probe methodology was designed to identify homozygous PSS-resistant, PSS-susceptible animals as well as heterozygous carriers. All cases genotyped by
luorescent hybridization probes were in agreement with PCR restriction enzyme digestion and sequencing
and showed a 100% concordance between the in vivo and in vitro porcine stress syndrome (PSS) susceptibility results.
conclusions and clinical relevance: The real-time PCR with luorescent hybridization probe method
described here provides a rapid, easily interpretable and reliable tool for genotyping the C1843T (Arg615Cys) polymorphism of the RYR1 gene. This new methodology may be useful in the wide-scale genotyping of
PSS-susceptibility and genetic selection.
Keywords: Malignant Hyperthermia, Caffeine-Halothane Contracture Test, Malignant Hyperthermia Diagnosis, Halothane Test, Succinilcholine Test, Porcine stress syndrome susceptibility, Arg615Cys, C1843T
SNP, molecular diagnostic, real-time PCR, luorescent hybridization probes
*These authors contributed equally to this work
(1)
(2)
(3)
(4)
(5)
(6)
(7)
Department of Physiology
IGUN, Faculty of Medicine
Faculty of Veterinary Medicine
Department of Pharmacy, Faculty of Sciences
CEIF, National University of Colombia, Bogotá, Colombia.
Department of Physiology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain.
[email protected]
Department of Botany and Genetics, Constantine the Philosopher University, Slovak Republic.
Recibido para publicación marzo 31 de 2008, Aceptado para publicación abril 9 de 2008
11
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Caminos J.E., Rojas J.E. Wilches M.A. y cols
inTRODUCTiOn
The Porcine Stress Syndrome (PSS) susceptibility
is a hypermetabolic syndrome involving skeletal
muscle disorder associated with a failure of the
calcium homeostasis in muscle ibers, characterized
by hyperthermia, muscle rigidity, and autonomic
dysfunction(1,2). Ryanodine receptor 1 (RYR1) and the
dihydropyridine receptor (DHPR) are involved in the
release of the calcium (Ca2+) stored in sarcoplasmic reticulum(3,4). PSS-susceptibility is inherited as
an autosomal recessive dysfunction that has been
clearly demonstrated and associated with a single
point mutation C1843T (Arg615Cys) in the Ca2+ release channel at the sarcoplasmic reticulum RYR1,
where the susceptible mutant allele is denominated
T and the wild-type - nonsusceptible allele C(5,6).
The frequency of the PSS gene in swine populations varies according to genetic breeds and country
of origin. Porcine stress syndrome has been reported
in different swine breeds and the incidence is higher
in lean, heavily muscled breeds such as Duroc, Large White, Pietrain, and Landrace. This susceptibility
trait was related to the incidence of acute stress and
sudden death, and an inferior quality meat generally
referred to as pale, soft, exudative, dark, irm and
dry, with back muscle necrosis(7).
Despite the number of laboratory tests developed
with the aim to identify PSS-susceptible animals or
carriers, none enables rapid diagnosis of PSS in an
acute situation and most of these lack the sensitivity
and speciicity to identify PSS-susceptible animals.
The in vitro contracture test remains the standard
test for the diagnosis of malignant hyperthermia
(MH) susceptibility in which live muscle specimens
are exposed to halothane and caffeine in the laboratory and the contracture response correlates with
elevated myoplasmic Ca2+ concentrations(8,9). In spite of the fact that the sensitivity of the in vitro contracture test is 97%, and the speciicity is approximately 85%, when this was used for subjects at high
risk(10), this methodology had limited application in
animals. This was because special laboratory facilities are required and the test must be run within
minutes after the specimen is obtained. Genotyping
must meet several key criteria, including speciicity,
sensitivity, fast turnaround, and cost-effectiveness.
Real -time PCR using luorescein-labeled hybridization probes fulils these criteria and furthermore is
a rapid, versatile, and cost-effective method for this
type of analysis(11).
The purpose of the present study was to develop a molecular genotyping method test by using a
hybridization probe and applying it to the analysis
12
of C1843T mutations of the RYR1 gene, which is
associated with the PSS susceptibility. Hybridization
probe methodology was designed to identify homozygous PSS-resistant, PSS-susceptible animals as
well as heterozygous carriers. Genotyping results
obtained by hybridization probe strategies were
conirmed by restriction analysis and sequencing. In
addition, phenotype/genotype correlation analyses
were developed by using the in vitro contracture test
and conirmed the in vivo halothane-succinylcholine
challenge. Our results showed that the simplicity
of real-time Polymerase Chain Reaction (PCR) technologies using luorescence quenching schemes,
allowed the diagnosis of the three genotypes of the
PSS. This new methodology is a tool that may contribute to genetic selection in order to obtain resistant
pigs to Porcine Stress Syndrome. The real-time PCR
with luorescent hybridization probes method described here provides a rapid, easily interpretable and
reliable tool for genotyping the C1843T (Arg615Cys)
polymorphism of the RYR1 gene.
ExPERiMEnTAL PROCEDURES
Three PSS-susceptible and PSS non-susceptible
crossbred swine races were used for the experiments: Pietrain X Landrace Belga, Pietrain X Large
White and Pietrain X Duroc. Swine were housed in
standard lighting and were given water and food ad
libitum until the beginning of the experiment, when
they were moved to a surgery room. All procedures
were in accordance to the Guide for the Care and Use
of Laboratory Animals (National Institute of Health),
and were approved by the Ethics Commission of
the Veterinary School at the National University of
Colombia.
Muscle samples biopsies, subjected to halothane-caffeine in vitro contracture tests were tested
according to the protocol of the North American
Malignant Hyperthermia Group protocol(8,12). Genotyping was done using a real-time PCR-based system
(LightCycler, Roche, and Mannheim, Germany) with
hybridization probes speciic for the RYR1 C1843T
SNP. Additionally, individual samples representing
the C/C, C/T or T/T genotypes analyzed by realtime PCR were also genotyped by DNA sequencing
and restriction fragment length polymorphism
techniques, proving the accuracy of the real-time
PCR method.
Initially, random samples of peripheral blood were
obtained from ninety-ive swine in a farm located
outside the metropolitan area of Bogotá, Colombia.
DNA samples plus controls (CC, CT and TT RYR1
genotyping, donated by Dr. R. Omelka, from the De-
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Diagnostic of Porcine Stress Syndrome Susceptibility
partment of Botany and Genetics, Constantine the
Philosopher University, Slovak Republic) were processed for screening the RYR1 C/T SNP (C1843T),
using polymerase chain reaction and restriction
fragment length polymorphism analysis with the
enzyme HhaI (Invitrogen), as described elsewhere(13).
For this purpose 5 mL of peripheral venous blood
was gathered from each swine by standard venepuncture and each blood sample was collected in
sterile tubes containing K3EDTA solution. The animals were genetically tested by standard methods
using genomic DNA isolated from blood leucocytes
(ChargeSwitch® gDNA Blood Kit, Invitrogen, USA),
according to the manufacturer's recommendations.
The purity and concentration of the extracted DNA
were determined by a UV–Vis spectrophotometer.
Twenty-one swine genotyping with the restriction
enzyme HhaI for the RYR1 C1843T SNP (CC n=9,
TC n=7, and TT n=5), were randomly selected for
the later development of the study.
Sequences for PCR primers and hybridization
probes used for SNP detection can be found in
Table 1. The DNA sequencing templates were PCR
ampliied from the Sus scrofa strain Yorkshire RYR1
gene (Accession M91456.1 [GenBank]) and primers
were designed using Primer3 software (http:// frodo.
wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The
primers ampliied a 329 bp fragment encompassing
the polymorphism C1843T.
The detection probe attached to the polymorphic
site and the nucleotides lanking it were speciic
for the T-allele (igure 1). Hybridization probes for
genotyping RYR1 gene whose mutation C1843T
(Arg615Cys) is associated with PSS -susceptibility,
were designed and obtained from Roche Molecular
The primer and Hhybridization probe sequences are
shown. Donor probe is labeled with luorescein at
the 3´, whilst the adjacent acceptor probe is labeled
with the speciic luorophore Red 640 (LC Red 640)
at the 5´ end region
Biochemical®. Acceptor probe were labeled at the
5'-end with LC Red 640 and phosphorylated on
the 3'-end by block extension. The donor probe
was labeled on the 3'-end with luorescein and was
designed to bind at a distance of three bases from
the acceptor probe.
The PCR reaction was carried out using the
LightCycler real-time PCR machine 2.0 (Roche
Diagnostics, Germany) and the software version
4.0. The reaction was performed in a volume of 20
μl with 2.5 mM MgCl2, 0.25 μM of both primers,
0.25 μM of both probes and 2 μl of 10 × LightCycler
® FastStart DNA Master HybProbe buffer (Roche
Diagnostics), and about 50 ng of template DNA. The
PCR protocol consisted of initial denaturation at 95
°C for 10 min, followed by 37 cycles of denaturation
(95 °C for 5 s, 20 °C/s), annealing (60 °C for 15 s, 20
°C/s) and elongation (72 °C for 20 s, 20 °C/s). This
was followed by melting curve analysis consisting
Figure 1: A representative fragment of the Sus scrofa strain Yorkshire RYR1 gene, showing the PCR primers and the luorophore-labeled hybridization probes for SNP C1843T. Probes designed to detect the SNP
C1843T were complementary to the antisense (reverse) strands of the RYR1. The SNP creates an A-T match
between the antisense strand and the donor probe the allele 1843T. Complete matching of donor probe to
the antisense strand results in a higher Tm of the mutant allele’s hybrid. The mismatch destabilizes the wild
type hybrid such that a decrease in the probe Tm occurs. P indicates the addition of a phosphate group.
13
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Caminos J.E., Rojas J.E. Wilches M.A. y cols
of 1 cycle at 95 °C for 30 s (20 °C/s), 40 °C for 20 s
(20 °C/s) and a temperature rise to 85 °C at a slope
of 0.2 °C/s with continuous measurement of luorescence. The genotyping results were interpreted
according to derivative melting curves [− d (F2 / F1)
/ dt] plotted against temperature.
To conirm the genotyping results obtained by
real-time PCR using hybridization probes strategies, restriction analysis and sequencing were used
(igure 2, 3). All PCR products were puriied using
Marligen's Gel Extraction Systems Kit (Marligen
Biosciences Inc.), according to the manufacturer's
recommendations. Sequencing was performed in
both directions by DYEnamic™ ET Dye Terminator
Kit (MegaBACE™) on a MegaBace DNA Analysis Systems (Amersham Biosciences). Sequencing results
were analyzed using nucleotide-nucleotide BLAST
2 SEQUENCES software ([GenBank]) to locate and
identify base changes. In addition, analyses of
DNA RYR1 amplicons were subjected to restriction
enzyme digestion during 4 hours, with the enzyme
HhaI (Invitrogen), according to the manufacturer's
recommendations. The digested DNA fragments
were visualized on a 1.5% agarose gel, with a 100bp DNA ladder (Invitrogen) as a molecular weight
marker, and stained with ethidium bromide (10 mg/
Figure 2: Derivative melting curve (-dF/dT) showing
the RYR1 single base genotyping (C/T). Samples are
wild type with a C-T mismatch to the hybridization
probe and a melting temperature of 54.2°C; the mutation with a perfect match (T) to the hybridization
probe and a melting temperature of 58.97°C, and
two heterozygous samples with both wild type and
mutant alleles. Melting analysis of a no template
control was also performed. The melting peaks
indicate that the RYR1 1843C wild type allele sequence has a lower Tm than the mutant 1843T allele
sequence due to homology between the probe and
mutant allele.
14
mL). Images of the gels were taken using Molecular
imaging Systems (Bio-Rad). In all assays, negative
and positive sample controls with known SNP C/T
genotypes were used.
To determine the associations between genetically tested swine for the RYR1 C1843T SNP and the
stress susceptibility phenotype, six PSS-susceptible
and PSS-nonsusceptible swine (C/C, n=2; C/T, n=2;
T/T, n=2; weight, 30 +/- 10 kg) were investigated.
In vivo studies with 3% halothane and in vitro contracture test from the gracilis muscle biopsy exposed
to halothane-caffeine were undertaken(12). Parallel
to the in vivo study, viable muscle specimens were
taken and exposed to in vitro contracture tests
following the protocol described by Marilyn Green
Larach and the North American Malignant Hyperthermia Group(8).
Figure 3: Melting Temperatures for Fluorescein
Probe/Allele. The homozygous mutant genotype
(TT) formed and perfect match T:A and were annealed to the probe at the luorescence acquisition temperature of 58.82 °C. The luorescein probe formed
a T:G mismatch with the wild-type sequence (CC),
lowering the Tm of the probe by 4.62°C from the
completely complementary duplex. Heterozygous
(CT) genotype had a melting peak at 58.97 °C (perfect match T:A) and other acquisition temperature
of 54.53 °C (T:G mismatch). Data were expressed
as means ± SEM (CC: n= 9; CT: n= 5; TT: n= 7), and
their statistical comparison was made by ANOVA
followed by the unpaired Student's t-test. A P value
of less than 0.05 was considered to be statistically
signiicant.
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Diagnostic of Porcine Stress Syndrome Susceptibility
RESULTS
A schematic representation of the adjacent luorescent hybridization probes used for genotyping the
RYR1 C1843T SNP is shown in igure 1. Table 1 lists
the sequences for PCR primers and hybridization
probes used for SNP detection. These probes were
designed so that during hybridization the donor
probe formed a mismatch with the wild-type allele
(C:T). The data collected during the melting phase
were used to genotype each sample and melting
peaks were obtained by plotting the luorescence
(F) versus temperature data as 2dF/dT.
Genotype analysis for the C1843T SNP is shown
in igure 2, where all genotypes are clearly distinguishable. The peak assignment is as given in igure
3. The G:T mismatch formed between the wild-type
allele with the luorescein probe, created a DTm of
4.62°C from the completely Watson-Crick paired
duplex (homozygous mutant allele T). A distinct
peak at 54.2 ± 0,10 °C is indicative of allele C1843,
whereas both the homozygous mutant allele (TT)
and the allele with the 1843T mutation from the
heterozygous samples, were annealed to the probe
at the luorescence acquisition temperature of 58,82
± 0,35°C (see igure 3).
Swine single point mutation C1843T (Arg615Cys) genotyping performed in the LightCycler
instrument was compared with conventional PCR
restriction fragment length analysis and sequence. PCR restriction fragment length analysis was
performed on all samples for the C1843T mutation with the enzyme HhaI (igure 4). Forward
and reverse primers were designed to produce a
329-bp-long amplicon (see table 1 and igure 4A).
The RYR1 C1843T mutation destroyed a HhaI
restriction site that upon restriction digestion of
the wild-type genomic DNA, yielded fragments of
239 and 90 bp (igure 4B). All cases genotyped by
luorescent hybridization probes agreed with PCR
restriction fragment length analysis. In addition,
results of genotyping with hybridization probes
and restriction analysis were compared with PCR
amplicon sequences and aligned to the Sus scrofa
strain Yorkshire RYR1 gene to verify melting curve
analyses (igure 5). Single base change at the targeted SNP site corresponded with results of melting
curve analyses (Figure 2). Direct DNA sequencing
analysis conirmed the different RYR1 genotypes
(igure 5) and revealed a C-to-T substitution that
results in an arginine-to-cysteine amino acid replacement at codon 615 (Arg615Cys). Variations from
wild-type to mutant alleles resulted in 4.62°C Tm
change (Figure 3).
Figure 4: Restriction analysis for the swine RYR1
C1843T SNP, determined by digestion with HhaI on
1.5% agarose gel electrophoresis stained with ethidium bromide. Allele speciic PCR for the C1843T
variant showing band patterns after screening: undigested 329-bp PCR product; wild-type (CC) genotype
restriction fragments of 239 and 90 bp; heterozygous
(CT) genotype restriction fragments of 329, 239 and
90 bp; and homozygous mutant (TT) genotype restriction fragment of 329 bp. MW, molecular weight
marker 100 bp.
In the current study, the correlation between
RYR1 C1843T genotype determination above by
different molecular approaches and the porcine
stress syndrome susceptibility has been investigated. Phenotype/genotype correlation analyses were
developed by using the in vitro contracture test and
conirmed by the in vivo halothane-succinylcholine
challenge (Figure 6A andB), as was described
elsewhere(12). All swine diagnosed as PSS-susceptible
by in vitro contracture test, were T allele homozygous
(TT) and heterozygous (CT) for the RYR1 mutation,
and all PSS non-susceptible swine were homozygous
for the C1843 allele (Figure 6). All results were in
agreement with contracture test parameters seen
with an increase in twitch height as Allen described
elsewhere(12). Furthermore, all swine diagnosed as
PSS-susceptible by the in vivo halothane-succinylcholine challenge were homozygous for the 1843T
allele whereas the wild-type (CC) and heterozygous
swine were PSS non-susceptible (data not shown).
These results conirm once again that autosomal
recessive Porcine Stress Syndrome susceptibility
disease is caused by the C1843T mutation in the
gene encoding the skeletal muscle calcium release
channel RYR1.
15
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Caminos J.E., Rojas J.E. Wilches M.A. y cols
Figure 5: Results of direct sequencing of a 329-bp
fragment of the RYR1 gene, showing the polymorphic
site at position: A) wild-type genotype (CC), B) heterozygous pattern (CT) and C) homozygous mutant (TT)
(indicated by arrows).
DiSCUSSiOn
Porcine stress syndrome (PSS) susceptibility,
has been associated with a single point mutation
C1843T (Arg615Cys) in the Ca2+ release channel
at the sarcoplasmic reticulum ryanodine receptor
1 (RYR1). The RYR1 gene is the best commercially
available marker used for predisposition of stress
susceptibility in several swine breeds. Changes in
16
Figure 6: The Caffeine–Halothane Contracture Test
in PSS-nonsusceptible and PSS-susceptible from the
swine gracilis muscle biopsy. (A) Normal (negative)
and (B) abnormal (positive) response after exposure
to 3% halothane. A positive halothane contracture test
is deined as a contracture greater than 0.2 – 0.7 g
after exposure to 3% halothane bubbled through the
tissue bath for 10 min. (C) Normal (negative) and (D)
abnormal (positive) response to caffeine. Caffeine
exposure is to 0.5, 1.0, 2.0, 4.0, 8.0, and 32 mM for 4
min in each concentration. A contracture greater than
0.3 g to 2 mM caffeine or less indicates susceptibility.
32 mM caffeine is added to the bath to determine
maximal response.
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Diagnostic of Porcine Stress Syndrome Susceptibility
the intracellular free calcium concentration are
relected in a variety of cellular responses, such as
muscle contraction, neuronal excitability, gene expression and metabolism variation. The functional
effect of the RYR1 Arg615Cys mutation (C1843T)
linked to neuromuscular disorders lead to a dysregulation of intracellular calcium homeostasis.
The mechanisms leading to the PSS-susceptibility
crisis involve a high concentration of free calcium
in the myoplasm released by a faulty membrane
system in calcium storing organelles including
sarcoplasmic reticulum(14). In the present study,
we used molecular and physiological (in vitro and
in vivo) approaches to determinate the PSS susceptibility.
Current methods for genotyping the C1843T
Porcine stress syndrome (PSS) susceptibility
mutations include allele-speciic oligonucleotide
hybridization and PCR restriction fragment length analysis, and these methods require multiple
manual steps and are time consuming(5,15). An
alternative is the use of luorescent hybridization
probes and rapid-cycle PCR, a technique that provides homogeneous ampliication and genotyping
in approximately 45 minutes. We have developed
a genotyping method by using a hybridization
probe and applied it to the analysis of C1843T
mutations of the RYR1 gene, associated with PSS
susceptibility.
Methods available for PSS- susceptibility genotyping are based on sequencing and restriction
fragment analysis, which requires several technical
steps. Although different PCR-based luorescent
techniques are being used for the diagnosis of
PSS-susceptibility none of them genotype by allele
/ hybridization probe Tm(16). In addition, the technology of luorescence resonance energy transfer
avoids additional steps, and the genotyping can
be done directly on extracted genomic DNA from
swine. Real time PCR using hybridization probe
systems based on luorescence resonance energy
transfer (FRET) have been established as effective
tools that allow the discrimination between closely
related sequences on the basis of a melting curve
analysis that confer very sensitive and highly speciic detection. This technology is useful for diagnostic purposes such as genotyping (SNP analysis)
and determination of genetic predisposition to
disease(11). In addition, Polymerase Chain Reaction ampliication and genotyping by luorescent
probe melting temperature, which reduces the
cost and invasiveness of sample collection, is a
rapid, versatile, and cost-effective method for this
type of analysis. Derivative melting curves for the
three genotypes of the RYR1 C1843T (Arg615Cys)
polymorphism are shown in Figure 2.
The thermal stability of a DNA duplex relies on
GC content, duplex length, and Watson-Crick base
pairing. Fluorescence from hybridization probes is
the direct result of the hybridization of two independent probes and this result in very high speciicity.
Changes from Watson-Crick pairing destabilize a
duplex by varying degrees depending on the length
of the mismatched duplex, the speciic mismatch,
the position of the mismatch, and neighboring base
pairs. The probe melting temperature is sequence
dependent, providing a simple method to genotype
mutations, including single base mutations(11). In this
study, the single base mismatch G:T (C1843T) in the
probe region decreases the melting temperature as
little as 4.62 °C in comparison with the completely
Watson-Crick paired duplex. The average melting
temperature was 54.2 ± 0.10 °C for the Wild-type
C allele and 58.82 ± 0,35°C for the mutant T allele.
The donor luorescein probe formed an G:T mismatch with the wild-type allele lowering the Tm of
the probe by 4.62 °C from the Watson-Crick paired
duplex, assuring a good separation of the C1843 and
1843T genotypes. The donor probe attached to the
polymorphic site was speciic for the T-allele. All cases
genotyped by luorescent hybridization probes were in
agreement with PCR restriction enzyme digestion and
sequencing in a random set of samples representing
all genotypes. When we validated the method with
positive DNA control generously donated by Dr. R.
Omelka (Department of Botany and Genetics, Constantine the Philosopher University, Slovak Republic),
the results matched completely.
Finally, we have demonstrated a 100% concordance between the in vivo and in vitro porcine stress
syndrome (PSS) susceptibility results and the ones
obtained by means of molecular genotyping such
as real-time PCR using luorescein-labeled hybridization probe, sequencing and restriction analysis.
The simplicity of real-time PCR technologies using
a luorescence quenching schemes, allowed diagnosis of the three genotypes of the PSS, and this
methodology is a tool that may contribute to the
genetic selection and to obtain pigs resistant to PSS.
In summary, this assay shows that real-time with
luorescent hybridization probes provide a rapid and
sensitive analysis for genotyping the RYR1 C1843T
(Arg615Cys) polymorphism and may be useful in the
wide-scale genotyping of PSS-susceptibility.
This new methodology is a tool that may contribute to the genetic selection and to obtain pigs
resistant to PSS and may be useful in the wide-scale
genotyping of PSS-susceptibility.
17
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Caminos J.E., Rojas J.E. Wilches M.A. y cols
AgRADECiMiEnTOS
Los autores agradecen al Dr. Henry Rosenberg,
Dra Sheila Muldoon, Dr. Saiid Bina y Dr. Gregory
Allen su decidida colaboración y apoyo en el desarrollo de este estudio.
de Investigación de la Universidad nacional de
Colombia en Bogotá, D.C. [email protected].
co, Teléfono: 3165000 ext. 15047
Dirección para correspondencia
Address correspondence to:
Professor Jorge Eduardo Caminos
Department of Physiology, Faculty of Medicine,
National University of Colombia, Bogotá, Colombia.
(E-mail: [email protected]).
Ciudad Universitaria - Bogotá D.C.- Colombia.
Ediicio "Uriel Gutiérrez" Transversal 38 N° 40-01
Phone (57)(1) 3165000 – ext 15047
Esta investigación se inanció con aportes del
Fondo de Investigaciones para Ciencias Básicas
de la Sociedad Colombiana de Anestesiología y
Reanimación soportado por la Revista Colombiana
de Anestesiología: email: [email protected].
co Fax: 6204619 Teléfono: 6196091 y la División
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